Name: technical applications of microelectrode array and patch clamp recordings on human induced pluripotent stem cell-derived cardiomyocytes
Uploaded: 2022-08-04T14:45:00
Description: Watch this Scientific Journal Video about Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

我们感谢Blake Wu校对稿件。这项工作得到了美国国立卫生研究院（NIH）R01 HL113006，R01 HL141371，R01 HL163680，R01 HL141851，U01FD005978和NASA NNX16A069A（JCW）以及AHA博士后奖学金872244（GMP）的支持。

1. 培养基和溶液的制备
<ol><li>通过混合 10 mL 瓶装 50x B27 补充剂和 500 mL RPMI 1640 培养基来制备 hiPSC-CM 维持培养基。将培养基储存在4°C并在一个月内使用。使用前将介质平衡至室温 （RT）。</li>
	<li>通过混合 20 mL 血清替代物和 180 mL hiPSC-CM 维持培养基（10% 稀释度，v/v）制备 hiPSC-CM 接种培养基。虽然新鲜制备的播种培养基是优选的，但它可以在4°C下储存不超过2周。使用前将培养基平衡...

<ol>
	<li>Wouters, O. J., McKee, M., Luyten, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimated+research+and+development+investment+needed+to+bring+a+new+medicine+to+market,+2009-2018.">Estimated research and development investment needed to bring a new medicine to market, 2009-2018.</a> Journal of the American Medical Association. 323 (9), 844-853 (2020).</li><li>Pang, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Workshop+report:+FDA+workshop+on+improving+cardiotoxicity+assessment+with+human-relevant+platforms.">Workshop report: FDA workshop on improving cardiotoxicity assessment with human-relevant platforms.</a> Circulation Research. 125 (9), 855-867 (2019).</li><li>Mamoshina, P., Rodriguez, B., Bueno-Orovio, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+broader+view+of+mechanisms+of+drug+cardiotoxicity.">Toward a broader view of mechanisms of drug cardiotoxicity.</a> Cell Reports Medicine. 2 (3), 100216 (2021).</li><li>Paik, D. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+induced+pluripotent+stem+cell-derived+cardiomyocytes+recapitulate+the+predilection+of+breast+cancer+patients+to+doxorubicin-induced+cardiotoxicity.">Human induced pluripotent stem cell-derived cardiomyocytes recapitulate the predilection of breast cancer patients to doxorubicin-induced cardiotoxicity.</a> Nature Medicine. 22 (5), 547-556 (2016).</li><li>Kitani, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-induced+pluripotent+stem+cell+model+of+trastuzumab-induced+cardiac+dysfunction+in+patients+with+breast+cancer.">Human-induced pluripotent stem cell model of trastuzumab-induced cardiac dysfunction in patients with breast cancer.</a> Circulation. 139 (21), 2451-2465 (2019).</li><li>Matsa, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptome+profiling+of+patient-specific+human+iPSC-cardiomyocytes+predicts+individual+drug+safety+and+efficacy+responses+in+vitro.">Transcriptome profiling of patient-specific human iPSC-cardiomyocytes predicts individual drug safety and efficacy responses in vitro.</a> Cell Stem Cell. 19 (3), 311-325 (2016).</li><li>Stillitano, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+susceptibility+to+drug-induced+long+QT+with+a+panel+of+subject-specific+induced+pluripotent+stem+cells.">Modeling susceptibility to drug-induced long QT with a panel of subject-specific induced pluripotent stem cells.</a> Elife. 6, (2017).</li><li>Zhang, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protocol+to+measure+contraction,+calcium,+and+action+potential+in+human-induced+pluripotent+stem+cell-derived+cardiomyocytes.">Protocol to measure contraction, calcium, and action potential in human-induced pluripotent stem cell-derived cardiomyocytes.</a> STAR Protocols. 2 (4), 100859 (2021).</li><li>Blinova, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=International+multisite+study+of+human-induced+pluripotent+stem+cell-derived+cardiomyocytes+for+drug+proarrhythmic+potential+assessment.">International multisite study of human-induced pluripotent stem cell-derived cardiomyocytes for drug proarrhythmic potential assessment.</a> Cell Reports. 24 (13), 3582-3592 (2018).</li><li>Greensmith, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca+analysis:+an+Excel+based+program+for+the+analysis+of+intracellular+calcium+transients+including+multiple,+simultaneous+regression+analysis.">Ca analysis: an Excel based program for the analysis of intracellular calcium transients including multiple, simultaneous regression analysis.</a> Computer Methods and Programs in Biomedicine. 113 (1), 241-250 (2014).</li><li>Ma, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determining+the+pathogenicity+of+a+genomic+variant+of+uncertain+significance+using+CRISPR/Cas9+and+human-induced+pluripotent+stem+cells.">Determining the pathogenicity of a genomic variant of uncertain significance using CRISPR/Cas9 and human-induced pluripotent stem cells.</a> Circulation. 138 (23), 2666-2681 (2018).</li><li>Lam, C. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identifying+the+transcriptome+signatures+of+calcium+channel+blockers+in+human+induced+pluripotent+stem+cell-derived+cardiomyocytes.">Identifying the transcriptome signatures of calcium channel blockers in human induced pluripotent stem cell-derived cardiomyocytes.</a> Circulation Research. 125 (2), 212-222 (2019).</li><li>Seeger, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+premature+termination+codon+mutation+in+MYBPC3+causes+hypertrophic+cardiomyopathy+via+chronic+activation+of+nonsense-mediated+decay.">A premature termination codon mutation in MYBPC3 causes hypertrophic cardiomyopathy via chronic activation of nonsense-mediated decay.</a> Circulation. 139 (6), 799-811 (2019).</li><li>Zhang, J. Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+cryopreservation+on+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+for+assessing+drug+safety+response+profiles.">Effects of cryopreservation on human induced pluripotent stem cell-derived cardiomyocytes for assessing drug safety response profiles.</a> Stem Cell Reports. 16 (1), 168-181 (2021).</li><li>Yu, B., Zhao, S. R., Yan, C. D., Zhang, M., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconvoluting+the+cells+of+the+human+heart+with+iPSC+technology:+cell+types,+protocols,+and+uses.">Deconvoluting the cells of the human heart with iPSC technology: cell types, protocols, and uses.</a> Current Cardiology Reports. 24 (5), 487-496 (2022).</li><li>Thomas, D., Cunningham, N. J., Shenoy, S., Wu, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human-induced+pluripotent+stem+cells+in+cardiovascular+research:+current+approaches+in+cardiac+differentiation,+maturation+strategies,+and+scalable+production.">Human-induced pluripotent stem cells in cardiovascular research: current approaches in cardiac differentiation, maturation strategies, and scalable production.</a> Cardiovascular Research. 118 (1), 20-36 (2022).</li><li>Zhu, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+developmental+cues+for+the+maturation+of+human+pluripotent+stem+cell-derived+cardiomyocytes.">Physical developmental cues for the maturation of human pluripotent stem cell-derived cardiomyocytes.</a> Stem Cell Research &amp; Therapy. 5 (5), 117 (2014).</li><li>Feyen, D. A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+maturation+media+improve+physiological+function+of+human+iPSC-derived+cardiomyocytes.">Metabolic maturation media improve physiological function of human iPSC-derived cardiomyocytes.</a> Cell Reports. 32 (3), 107925 (2020).</li><li>Gintant, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+human+induced+pluripotent+stem+cell-derived+cardiomyocytes+in+preclinical+cancer+drug+cardiotoxicity+testing:+a+scientific+statement+from+the+American+Heart+Association.">Use of human induced pluripotent stem cell-derived cardiomyocytes in preclinical cancer drug cardiotoxicity testing: a scientific statement from the American Heart Association.</a> Circulation Research. 125 (10), 75-92 (2019).</li><li>Kussauer, S., David, R., Lemcke, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hiPSCs+Derived+cardiac+cells+for+drug+and+toxicity+screening+and+disease+modeling:+what+micro-+electrode-array+analyses+can+tell+us.">hiPSCs Derived cardiac cells for drug and toxicity screening and disease modeling: what micro- electrode-array analyses can tell us.</a> Cells. 8 (11), (2019).</li><li>Ng, C. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+discovery+of+trafficking-deficient+variants+in+the+cardiac+potassium+channel+KV11.1.">High-throughput discovery of trafficking-deficient variants in the cardiac potassium channel KV11.1.</a> Heart Rhythm. 17 (12), 2180-2189 (2020).</li><li>Heyne, H. 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人类iPSC技术已成为疾病建模和药物筛选的强大平台。在这里，我们描述了用于测量hiPSC-CM收缩力，场电位，动作电位和Ca2 +瞬态的详细协议。该协议提供了hiPSC-CM收缩力和电生理学的全面表征。这些功能测定已应用于我们组12，13，18，24，25，26，<sup cl...

药物开发是一个漫长而昂贵的过程。一项针对美国食品和药物管理局 （FDA） 在 2009 年至 2018 年间批准的新治疗药物的研究报告称，资本化研究和临床试验的估计成本中位数为每种产品 9.85 亿美元1。药物引起的心脏毒性是药物损耗和退出市场的主要原因2.值得注意的是，多类治疗药物中报告了心脏毒性3。因此，心脏安全性评估是药物开发过程中的关键组成部分。目前的心脏安全性评估范式仍然高度依赖于动物模型。然而，与使用动物模型的物种差异越来越被认为是人类患者药物诱导的心脏毒性预测不准确的主要原因4。例如，由于不同复极电流的贡献，人类和小鼠之间的心脏动作电位形态差异很大5。此外，可影响心脏生理学的心肌球蛋白和环状RNA的差异亚型已在物种6，7中得到充分记录。为了弥合这些差距，必须建立一个可靠、高效和基于人的临床前心脏安全性评估模型。
诱导多能干细胞（iPSC）技术的突破性发明产生了前所未有的药物筛选和疾病建模平台。在过去的十年中，产生人类诱导的多能干细胞衍生心肌细胞（hiPSC-CMs）的方法已经非常成熟8，9。hiPSC-CMs在疾病建模、药物诱导的心脏毒性筛查和精准医学中的潜在应用引起了极大的兴趣。例如，hiPSC-CMs已被用于模拟由遗传引起的心脏病的病理表型，例如长QT综合征10，肥厚型心肌病11，12和扩张型心肌病13，14，15。因此，已经确定了与心脏病发病机制有关的关键信号通路，这可以阐明有效治疗的潜在治疗策略。此外，hiPSC-CMs已被用于筛选与抗癌药物相关的药物诱导的心脏毒性，包括多柔比星，曲妥珠单抗和酪氨酸激酶抑制剂16，17，18;减轻由此产生的心脏毒性的策略正在研究中。最后，hiPSC-CMs中保留的遗传信息允许在个体和人群水平上筛选和预测药物诱导的心脏毒性19，20。总的来说，hiPSC-CMs已被证明是个性化心脏安全预测的宝贵工具。
该协议的总体目标是建立全面有效地研究hiPSC-CMs功能特征的方法，这对于将hiPSC-CMs应用于疾病建模，药物诱导的心脏毒性筛查和精准医学非常重要。在这里，我们详细介绍了一系列功能测定来评估hiPSC-CMs的功能特性，包括收缩力，场电位，动作电位和钙（Ca2+）处理的测量（图1）。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>35 mm glass bottom dish with 20 mm micro-well #1.5 cover glass</td>
			<td>Cellvis</td>
			<td>D35-20-1.5-N</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>50x B27 supplements</td>
			<td>Life Technologies</td>
			<td>17504-044</td>
			<td>hiPSC-CM culture medium</td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>E &#38; K Scientific</td>
			<td>EK-27160</td>
			<td>hiPSC-CM culture</td>
		</tr>
		<tr>
			<td>96-well flat clear bottom black polystyrene TC-treated microplates</td>
			<td>Corning</td>
			<td>3603</td>
			<td>Contraction motion measurement</td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Sigma-Aldrich</td>
			<td>A6964</td>
			<td>Enzymatic dissociation</td>
		</tr>
		<tr>
			<td>Axion&#39;s Integrated Studio (AxIS)</td>
			<td>Axion Biosystems</td>
			<td></td>
			<td>navigator software</td>
		</tr>
		<tr>
			<td>Borosilicate glass capillaries</td>
			<td>Harvard Apparatus</td>
			<td>BF 100-50-10,</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>CaCl2 1 M in H2O</td>
			<td>Sigma-Aldrich</td>
			<td>21115</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>Cell counting chamber slides</td>
			<td>ThermoFisher Scientific</td>
			<td>C10228</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>CytoView 48-well MEA plates</td>
			<td>Axion Biosystems</td>
			<td>M768-tMEA-48B</td>
			<td>MEA</td>
		</tr>
		<tr>
			<td>DMEM/F12</td>
			<td>Gibco/Life Technologies</td>
			<td>12634028</td>
			<td>Extracellular matrix medium</td>
		</tr>
		<tr>
			<td>DPBS, no calcium, no magnesium</td>
			<td>Fisher Scientific</td>
			<td>14-190-250</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>EPC 10 USB&#160;patch clamp amplifier</td>
			<td>Warner Instruments</td>
			<td>89-5000</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>Fura-2, AM, cell permeant</td>
			<td>ThermoFisher Scientific</td>
			<td>F1221</td>
			<td>Ca2+ transient measurement</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>hiPSCs</td>
			<td>Stanford Cardiovascular Institute iPSC Biobank</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Sigma-Aldrich</td>
			<td>529552</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>KnockOut Serum Replacement</td>
			<td>ThermoFisher Scientific</td>
			<td>10828-028</td>
			<td>hiPSC-CM seeding medium</td>
		</tr>
		<tr>
			<td>KOH 8&#160;M</td>
			<td>Sigma-Aldrich</td>
			<td>P4494</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>Lambda DG 4</td>
			<td>Sutter Instrument Company</td>
			<td></td>
			<td>Ca2+ transient measurement; ultra-high-speed wavelength switching light source</td>
		</tr>
		<tr>
			<td>Luna-FL automated fluorescence cell counter</td>
			<td>WISBIOMED</td>
			<td>LB-L20001</td>
			<td>Cell counting</td>
		</tr>
		<tr>
			<td>Maestro Pro&#160;MEA system</td>
			<td>Axion Biosystems</td>
			<td></td>
			<td>MEA</td>
		</tr>
		<tr>
			<td>Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td>Extracellular matrix medium</td>
		</tr>
		<tr>
			<td>MgATP</td>
			<td>Sigma-Aldrich</td>
			<td>A9187</td>
			<td>Intracellular pipette solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>S9888</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NaOH 10&#160;M</td>
			<td>Sigma-Aldrich</td>
			<td>72068</td>
			<td>Tyrode&#8217;s solution</td>
		</tr>
		<tr>
			<td>NIS Elements AR</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127 (20% Solution in DMSO)</td>
			<td>ThermoFisher Scientific</td>
			<td>P3000MP</td>
			<td>Ca2+ transient measurement</td>
		</tr>
		<tr>
			<td>RPMI 1640 medium</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>hiPSC-CM culture medium</td>
		</tr>
		<tr>
			<td>Sony SI8000 Cell Motion Imaging System</td>
			<td>Sony Biotechnology</td>
			<td></td>
			<td>Contraction motion measurement</td>
		</tr>
		<tr>
			<td>Sutter Micropipette puller</td>
			<td>Sutter Instruments</td>
			<td>P-97</td>
			<td>Patch clamp</td>
		</tr>
		<tr>
			<td>Trypan blue stain</td>
			<td>Life Technologies</td>
			<td>T10282</td>
			<td>Cell counting</td>
		</tr>
	</tbody>
</table>

technical applications of microelectrode array and patch clamp recordings on human induced pluripotent stem cell-derived cardiomyocytes

药物引起的心脏毒性是药物损耗和退出市场的主要原因。因此，使用适当的临床前心脏安全性评估模型是药物开发过程中的关键步骤。目前，心脏安全性评估仍然高度依赖于动物研究。然而，由于物种特异性差异，特别是在心脏电生理特性方面，动物模型受到人类翻译特异性差的困扰。因此，迫切需要开发一种可靠、高效和基于人的临床前心脏安全性评估模型。人诱导的多能干细胞来源心肌细胞（hiPSC-CMs）已成为药物诱导的心脏毒性筛查和疾病建模的 宝贵体外 模型。hiPSC-CMs可以从具有不同遗传背景和各种疾病状况的个体中获得，使其成为单独评估药物诱导的心脏毒性的理想替代品。因此，需要建立全面研究hiPSC-CMs功能特征的方法。在该协议中，我们详细介绍了可以在hiPSC-CMs上评估的各种功能测定，包括收缩力，场电位，动作电位和钙处理的测量。总体而言，将hiPSC-CMs纳入临床前心脏安全性评估有可能彻底改变药物开发。

该协议描述了如何测量hiPSC-CM的收缩运动，场电位，动作电位和Ca2 + 瞬态。包括酶消化、细胞接种、维持和功能测定传导的示意图如图 1 所示。hiPSC-CM单层的形成对于收缩运动测量是必要的（图2B）。hiPSC-CMs收缩-松弛运动的代表性迹线如图 2C所示。分析仪软件通过使用默认设置检测收缩开始、收缩峰值、收缩结束、松弛峰?...

Watch this Scientific Journal Video about Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes at JoVE.com

Technical Applications of Microelectrode Array and Patch Clamp Recordings on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes

人诱导的多能干细胞来源的心肌细胞（hiPSC-CMs）已成为药物诱导的心脏毒性筛查和疾病建模的有前途的 体外 模型。在这里，我们详细介绍了用于测量hiPSC-CMs的收缩力和电生理学的方案。

微电极阵列和膜片钳记录在人诱导多能干细胞来源心肌细胞上的技术应用

Drug-induced cardiotoxicity is the leading cause of drug attrition and withdrawal from the market. Therefore, using appropriate preclinical cardiac safety ...

心脏安全性评估是药物开发过程中的关键组成部分。人iPSC来源的心肌细胞已被证明是一种可靠、高效和基于人类的临床前心脏安全性评估模型。我们已经建立了全面研究人iPSC来源心肌细胞功能特征的方法，这些方法具有非常重要的疾病建模，药物诱导的心脏毒性筛查和精准医学。越来越多地将人iPSC来源的心肌细胞纳入标准的临床前安全性评估过程，有可能提高候选化合物毒性预测的准确性。首先培养人iPSC来源的心肌细胞至少10天，然后再进行测量。打开温度控制器并将其设置为 37 摄氏度。打开显微镜、相机和二氧化碳供应。用适量的无菌去离子水填充板架的水套。将96孔板放在板架上，并将细胞孵育至少五分钟。打开视图软件，然后将相机帧速率设置为 75 帧/秒，并将视频或图像分辨率设置为 1024 x 1024 像素。应用自动对焦和自动亮度功能，记录人iPSC来源心肌细胞的视频和图像。小心地将8微升的额外细胞基质涂层溶液滴放在48孔微电极阵列板的每个孔的记录电极区域上。然后在孔周围区域加入三毫升无菌去离子水，以防止涂层溶液蒸发。使用200微升移液器吸头，在同一行或同一列内从孔表面去除大部分额外的细胞基质培养基，而不接触电极。在记录电极区域上方的8微升接种培养基液滴中每孔接种50，000个细胞。重复直到所有孔都接种了细胞。然后将微电极阵列板在加湿的细胞培养箱中孵育。向微电极阵列板的每个孔中缓慢加入150微升种子培养基。在电镀后第10天，在显微镜下检查自发跳动的人iPSC CM单层的密度。将48孔微电极阵列板放入记录仪器中。确保温度为37摄氏度，二氧化碳为5%打开导航器软件。在实验设置窗口中，选择心脏实时配置和场电位记录。应用自发或糊状跳动配置。在拍频检测参数中，将检测阈值设置为 300 微伏，最小跳动周期为 250 毫秒，最大跳动周期为 5 秒。选择多项式回归进行场势持续时间计算。检查基线电活动信号是否成熟稳定，获取基线心脏活动1-3分钟。电镀后10天进行记录。用tyrode溶液替换人iPSC CM维持培养基，并让细胞适应15分钟。将温度传感器插入腔室，然后将 35 毫米的培养皿放入其中。将温度调节到37摄氏度。使用微量移液器拉拔器从硼硅酸盐玻璃毛细管中拉出微量移液器。用细胞内溶液填充微量移液器，并将它们插入连接到膜片钳放大器头部级的支架中。打开采集软件，加载动作电位记录协议，选择电压钳位配置。将微量移液器插入浴液中，并选择合适的单个人iPSC CM。 使用显微操纵器调整并降低微量移液器在所选细胞旁边的位置。当移液器吸头插入浴液中时，在示波器监视器上检查移液器电阻。将移液器靠近细胞放置，电流脉冲将略微减小，以反映增加的密封电阻。用负压轻轻抽吸以增加阻力。这将形成一个千兆印章。然后应用密封功能。施加额外的吸力以破坏膜以获得整个细胞记录配置。此时，使用放大器对话框从电压箝位模式切换到电流箝位模式，或者不注入电流。按记录按钮生成并保存动作电位记录文件。在电镀后至少10天进行钙瞬时测量。准备新鲜的泰罗德溶液和Fura-2 AM装载溶液。吸出人iPSC CM维持培养基，并用tyrode溶液清洗腔室两次。应用100微升Fura-2AM负载溶液，并在室温下孵育10分钟。用tyrode溶液替换Fura-2 AM上样溶液，并孵育至少五分钟以使Fura-2 AM完全脱酯。在倒置落射荧光显微镜的40倍油浸物镜上加入一滴浸油。将细胞室放在显微镜的载物台适配器中。将温度控制器电线安装到浴室室，并将其设置为37摄氏度。安装电场刺激电极，并将脉冲设置为 0.5 赫兹，持续时间为 20 毫秒。开启超高速波长切换光源，设置为340纳米和380纳米快速切换模式。对两个波长应用20毫秒的曝光时间，并应用20秒的记录时间。录制反映钙瞬态实时变化的视频，将数据导出到电子表格进行分析。人iPSC来源的心肌细胞单层用于收缩运动测量。使用分析仪软件分析收缩松弛运动的轨迹，检测到收缩开始、收缩峰值、收缩结束、松弛峰值和松弛结束。在微电极阵列板内的所有电极上观察到人iPSC来源的心肌细胞单层的完全覆盖。每个电极记录的成熟波形反映了具有可识别特征的心野电位。经过分析，得到了跳频周期、场电位持续时间和尖峰幅度。电流钳模式下的全细胞膜片钳记录记录单个心肌细胞的自发动作电位。经过分析，得到了振幅、最大舒张电位和动作电位持续时间等多个参数。经过钙成像分析，得到F340随时间推移的F380比值，并绘制了受刺激钙瞬变的原始痕迹。此外，还获得了振幅、舒张期钙和钙衰变tau等参数。重要的是使用处于良好跳动条件下的人iPSC来源的心肌细胞，以及确保人iPSC来源的心肌细胞的高纯度。该协议包括四种特定技术，用于研究人类iPSC衍生的心肌细胞功能，其他配置或自动是否研究在心脏中起关键作用的心脏离子电流

Research

JoVE Journal

Medicine

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

人类前列腺上皮细胞形成的使用鼠类泌尿生殖窦间质细胞和人类干细胞的组织重组

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

科研

医学

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

隔离与人类心室肌细胞的功能研究从新鲜手术样品

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

Tissue-simulating Phantoms for Assessing Potential Near-infrared Fluorescence Imaging Applications in Breast Cancer Surgery

组织鬼怪模拟，以评估乳癌手术潜在的近红外荧光成像中的应用

Inaccuracies in intraoperative tumor localization and evaluation of surgical margin status result in suboptimal outcome of breast-conserving surgery ...

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

人类癫痫术后皮层组织的多电极阵列录音

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

从生成性肌营养不良患者诱导多能干细胞的：尿源性细胞的有效整合，免费重编程

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

Rapid and Efficient Generation of Recombinant Human Pluripotent Stem Cells by Recombinase-mediated Cassette Exchange in the AAVS1 Locus

通过重组酶介导的盒式交换在重组人多能干细胞的快速高效生成<em&gt; AAVS1</em&gt;轨迹

Even with the revolution of gene-targeting technologies led by CRISPR-Cas9, genetic modification of human pluripotent stem cells&#160;(hPSCs)&#160;is ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

利用外束辐射对小型动物模型的多能干细胞衍生畸胎瘤进行靶向选择性治疗

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

Sarcomere Shortening of Pluripotent Stem Cell-Derived Cardiomyocytes using Fluorescent-Tagged Sarcomere Proteins.

使用荧光标记肉瘤蛋白缩短多能干细胞衍生心肌细胞的肉瘤。

Pluripotent stem cell-derived cardiomyocytes (PSC-CMs) can be produced from both embryonic and induced pluripotent stem (ES/iPS) cells. These cells ...

Directed Induction of Retinal Organoids from Human Pluripotent Stem Cells

从人多能干细胞中定向诱导视网膜类器官

Retinal cell transplantation is a promising therapeutic approach, which could restore the retinal architecture and stabilize or even improve the visual ...

Microelectrode Array Recording of Sinoatrial Node Firing Rate to Identify Intrinsic Cardiac Pacemaking Defects in Mice

微电极阵列记录中天节点发射率，以识别小鼠内在心脏起搏缺陷

The sinoatrial node (SAN), located in the right atrium, contains the pacemaker cells of the heart, and dysfunction of this region can cause tachycardia or ...